Osteosarcoma (OSA) occurs in both children/young adults and in dogs, with an annual incidence of 450 and 25,000 cases, respectively, making dogs a useful comparative oncology model for the development of OSA treatments. Unfortunately, outcomes for patients with OSA have plateaued over the past three decades. Thus, new treatment approaches beyond the standard of care (SOC) surgery and chemotherapy are sorely needed. As a novel therapy for OSA, our group is developing Magnetic Resonance Imaging (MRI)-guided cryotherapy in conjunction with image-guided intratumoral injection of an immune adjuvant. To this end, we will perform a canine clinical trial comparing MRI-guided cryotherapy alone or in conjunction with intratumoral immune adjuvant administration in spontaneously occurring, non-metastatic, canine appendicular OSA. The proposed immune correlative studies will provide crucial insights into the mechanisms of immune regulation in OSA tumors and the immunologic impact of the therapies to guide future improvements. While cryotherapy directly kills OSA tumor cells, our intent is to activate the patient?s adaptive immune system against tumor antigens that are presented from the dying tumor cells, with the addition of intratumoral delivery of an immune adjuvant. Following local control surgery, we hypothesize that the primed adaptive immune response can delay or prevent the occurrence of metastatic disease, which is uniformly fatal. Using MRI, one can precisely determine the extent of tumor tissue that is frozen and killed by cryotherapy without exposure to ionizing radiation. Others have demonstrated an influx of macrophage and myeloid cells following cryoablation. Via intratumoral injection, we will deliver a novel Stimulator of Interferon Gene (STING) agonist following cryotherapy to activate the infiltrating innate immune cells. STING pathway activation results in extensive cytokine induction, the activation of antigen presenting cells, and subsequent priming of T cells. Our specific aims are: 1. To determine whether MRI-guided cryoablation of OSA generates an immune response based on cytokine assays, immunohistochemistry (IHC), and immunoassays that can prevent metastatic progression, as assessed by non-invasive imaging and thereby increase overall survival compared to SOC treatment. 2. To characterize the immune response and safety of CT-guided STING agonist intratumoral administration in canine OSA. 3. To determine whether MRI-guided cryoablation combined with CT-guided intratumoral STING agonist administration prevents metastatic disease progression and prolongs survival compared with SOC treatment for OSA. The high incidence of OSA in dogs with a high rate of metastatic disease provides an ideal animal model with which to study this novel image-guided treatment of OSA using clinically relevant imaging systems with the potential for rapid translation and application in other solid tumors.